Abstract
Cryptic species are commonly misidentified because of high morphological similarities to other species. One group of plants that may harbor large numbers of cryptic species is the quillworts (Isoëtes spp.), an ancient aquatic plant lineage. Although over 350 species of Isoëtes have been reported globally, only ten species have been recorded in China. The aim of this study is to better understand Isoëtes species diversity in China. For this purpose, we systematically explored the phylogeny and evolution of Isoëtes using complete chloroplast genome (plastome) data, spore morphology, chromosome number, genetic structure, and haplotypes of almost all Chinese Isoëtes populations. We identified three ploidy levels of Isoëtes in China-diploid (2n = 22), tetraploid (2n = 44), and hexaploid (2n = 66). We also found four megaspore and microspore ornamentation types in diploids, six in tetraploids, and three in hexaploids. Phylogenetic analyses confirmed that I. hypsophila as the ancestral group of the genus and revealed that Isoëtes diploids, tetraploids, and hexaploids do not form monophyletic clades. Most individual species possess a single genetic structure; however, several samples have conflicting positions on the phylogenetic tree based on SNPs and the tree based on plastome data. All 36 samples shared 22 haplotypes. Divergence time analysis showed that I. hypsophila diverged in the early Eocene (∼48.05 Ma), and most other Isoëtes species diverged 3-20 Ma. Additionally, different species of Isoëtes were found to inhabit different water systems and environments along the Yangtze River. These findings provide new insights into the relationships among Isoëtes species in China, where highly similar morphologic populations may harbor many cryptic species.